R Notebook

Install

library(BSgenome.Hsapiens.UCSC.hg19)
#library(chromVAR)
fragment.counts.sigs <- addGCBias(fragment.counts.sigs, 
   genome = BSgenome.Hsapiens.UCSC.hg19)

Load packages

##try

Trying to integrate everything

#function that creates list of the sample path of the file h5
make.sample.path <- function(pathh){
  sample.path=list()
  
                sample.path <-list.files(path=pathh,
                      pattern="h5", recursive = T, full.names = TRUE) 
               
                return(sample.path)
        
}

make.fragpath <- function(pathh){
  sample.path<-list()
  
                sample.path <-list.files(path=pathh,
                      pattern=".tsv.gz$", recursive = T, full.names = TRUE) 
               
                return(sample.path)
        
}
#function that creates list of all the multiome h5 files read by Read10x for each sample 

make.multiome.Listas <- function(path){
  sample.name <-list()
   sample.pathList <-list()
   pbmc_list <-list()
   fragpathList<-list()
  # get gene annotations for hg38

  
                sample.pathList<- make.sample.path(path)
                fragpathList<- make.fragpath(path)
                sample.name<- sub(".*Experiment\\/(.*)\\/filtered_feature_bc_matrix.h5","\\1", sample.pathList, perl = TRUE )
                 annotation <- GetGRangesFromEnsDb(ensdb = EnsDb.Hsapiens.v86)
                 seqlevelsStyle(annotation) <- "UCSC"
                 genome(annotation) <- "hg38"
                 
                  for (i in seq_along(sample.pathList)){
                  counts = Read10X_h5(filename=sample.pathList[[i]])
                  fragpath <-fragpathList[[i]]
                  #print(fragpath)
                  pbmc<-CreateSeuratObject(counts = counts$`Gene Expression`, assay = "RNA")
                  pbmc[["ATAC"]] <- CreateChromatinAssay(
  counts = counts$Peaks,
  sep = c(":", "-"),
  fragments = fragpath,
  annotation = annotation
)
                  pbmc_list<-c(pbmc_list,pbmc)
                 } 
                 return(pbmc_list)
    }
  
        
#Create a multiome list of all the Seurat Object with Signac Cromatine assay

m<- make.multiome.Listas("data/Experiment")

Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
Fetching data...OK
Parsing exons...OK
Defining introns...OK
Defining UTRs...OK
Defining CDS...OK
aggregating...
Done
The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)'giveCsparse' has been deprecated; setting 'repr = "T"' for youGenome matrix has multiple modalities, returning a list of matrices for this genome
Computing hash
Checking for 5788 cell barcodes
'giveCsparse' has been deprecated; setting 'repr = "T"' for youGenome matrix has multiple modalities, returning a list of matrices for this genome
Computing hash
Checking for 7460 cell barcodes
'giveCsparse' has been deprecated; setting 'repr = "T"' for youGenome matrix has multiple modalities, returning a list of matrices for this genome
Computing hash
Checking for 5572 cell barcodes
'giveCsparse' has been deprecated; setting 'repr = "T"' for youGenome matrix has multiple modalities, returning a list of matrices for this genome
Computing hash
Checking for 10837 cell barcodes
'giveCsparse' has been deprecated; setting 'repr = "T"' for youGenome matrix has multiple modalities, returning a list of matrices for this genome
Computing hash
Checking for 6919 cell barcodes
'giveCsparse' has been deprecated; setting 'repr = "T"' for youGenome matrix has multiple modalities, returning a list of matrices for this genome
Computing hash
Checking for 5530 cell barcodes
'giveCsparse' has been deprecated; setting 'repr = "T"' for youGenome matrix has multiple modalities, returning a list of matrices for this genome
Computing hash
Checking for 7910 cell barcodes
'giveCsparse' has been deprecated; setting 'repr = "T"' for youGenome matrix has multiple modalities, returning a list of matrices for this genome
Computing hash
Checking for 5845 cell barcodes
'giveCsparse' has been deprecated; setting 'repr = "T"' for youGenome matrix has multiple modalities, returning a list of matrices for this genome
Computing hash
Checking for 7181 cell barcodes
'giveCsparse' has been deprecated; setting 'repr = "T"' for youGenome matrix has multiple modalities, returning a list of matrices for this genome
Computing hash
Checking for 6103 cell barcodes
'giveCsparse' has been deprecated; setting 'repr = "T"' for youGenome matrix has multiple modalities, returning a list of matrices for this genome
Computing hash
Checking for 7861 cell barcodes

Quality control

VlPlot_Filtering <- function(multiomeList){
  pbmc_list <-list()
  
  for (i in multiomeList){ #for each object in the multiome List. 
  
  DefaultAssay(i) <- "ATAC"

i <- NucleosomeSignal(i)
i <- TSSEnrichment(i)

print(VlnPlot(
  object = i,
  features = c("nCount_RNA", "nCount_ATAC", "TSS.enrichment", "nucleosome_signal"),
  ncol = 4,
  pt.size = 0))
pbmc_list<-c(pbmc_list,i)
                 } 
                 return(pbmc_list)
}

pbmc<-VlPlot_Filtering(m)

Found 5788 cell barcodes

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Done Processing 28 million linesExtracting TSS positions
Extracting fragments at TSSs

  |                                                  | 0 % ~calculating  

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |+++++                                             | 10% ~03m 11s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |++++++++++                                        | 20% ~03m 03s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |+++++++++++++++                                   | 30% ~02m 50s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++                              | 40% ~02m 19s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |+++++++++++++++++++++++++                         | 50% ~01m 55s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++++++++++++                    | 60% ~01m 35s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 13s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++++++++++++++++++++++          | 80% ~47s          

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~24s          

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=04m 01s

Computing TSS enrichment score
Found 7460 cell barcodes

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  |                                                  | 0 % ~calculating  
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  |++++++++++++++++++++++++++++++++++++++++          | 80% ~41s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~01m 03s      
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=09m 49s

Found 5572 cell barcodes

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  |                                                  | 0 % ~calculating  
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  |+++++++++++++++                                   | 30% ~02m 39s      
  |++++++++++++++++++++                              | 40% ~02m 13s      
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  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 13s      
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~48s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~25s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 60s

Found 10837 cell barcodes

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  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~02m 29s      
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  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~47s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=07m 25s

Found 6919 cell barcodes

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  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~02m 19s      
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  |++++++++++++++++++++++++++++++++++++++++          | 80% ~49s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~24s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=04m 03s

Found 5530 cell barcodes

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  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~03m 01s      
  |++++++++++                                        | 20% ~02m 33s      
  |+++++++++++++++                                   | 30% ~02m 27s      
  |++++++++++++++++++++                              | 40% ~02m 07s      
  |+++++++++++++++++++++++++                         | 50% ~01m 43s      
  |++++++++++++++++++++++++++++++                    | 60% ~01m 21s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 03s      
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~42s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~21s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 34s

Found 7910 cell barcodes

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  |                                                  | 0 % ~calculating  
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  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 04s      
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~42s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~21s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 30s

Found 5845 cell barcodes

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  |                                                  | 0 % ~calculating  

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |+++++                                             | 10% ~02m 41s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |++++++++++                                        | 20% ~02m 51s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |+++++++++++++++                                   | 30% ~02m 28s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++                              | 40% ~02m 11s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |+++++++++++++++++++++++++                         | 50% ~01m 54s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++++++++++++                    | 60% ~01m 30s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 13s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++++++++++++++++++++++          | 80% ~48s          

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~24s          

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=04m 04s

Found 7181 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines
Done Processing 28 million lines
Done Processing 29 million lines
                                                  
Done Processing 30 million lines
Done Processing 31 million lines
Done Processing 32 million lines
Done Processing 33 million lines
Done Processing 34 million lines
Done Processing 35 million lines

  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~02m 36s      
  |++++++++++                                        | 20% ~02m 22s      
  |+++++++++++++++                                   | 30% ~02m 18s      
  |++++++++++++++++++++                              | 40% ~02m 01s      
  |+++++++++++++++++++++++++                         | 50% ~01m 41s      
  |++++++++++++++++++++++++++++++                    | 60% ~01m 23s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 02s      
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~43s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~21s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 34s

Found 6103 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines
Done Processing 28 million lines
Done Processing 29 million lines
                                                  
Done Processing 30 million lines

  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~02m 34s      
  |++++++++++                                        | 20% ~02m 24s      
  |+++++++++++++++                                   | 30% ~02m 26s      
  |++++++++++++++++++++                              | 40% ~01m 59s      
  |+++++++++++++++++++++++++                         | 50% ~01m 37s      
  |++++++++++++++++++++++++++++++                    | 60% ~01m 25s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 03s      
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~42s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~21s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 25s

Found 7861 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines
Done Processing 28 million lines
Done Processing 29 million lines
                                                  
Done Processing 30 million lines
Done Processing 31 million lines
Done Processing 32 million lines
Done Processing 33 million lines
Done Processing 34 million lines
Done Processing 35 million lines
Done Processing 36 million lines
Done Processing 37 million lines
Done Processing 38 million lines
Done Processing 39 million lines

  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~02m 23s      
  |++++++++++                                        | 20% ~02m 56s      
  |+++++++++++++++                                   | 30% ~02m 35s      
  |++++++++++++++++++++                              | 40% ~02m 21s      
  |+++++++++++++++++++++++++                         | 50% ~01m 52s      
  |++++++++++++++++++++++++++++++                    | 60% ~01m 28s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 09s      
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~45s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~22s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 43s

pbmc<-VlPlot_Filtering(m)

Found 5788 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines
Done Processing 28 million linesExtracting TSS positions
Extracting fragments at TSSs

  |                                                  | 0 % ~calculating  

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |+++++                                             | 10% ~02m 37s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |++++++++++                                        | 20% ~02m 18s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |+++++++++++++++                                   | 30% ~02m 13s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++                              | 40% ~01m 58s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |+++++++++++++++++++++++++                         | 50% ~01m 44s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++++++++++++                    | 60% ~01m 25s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 03s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++++++++++++++++++++++          | 80% ~45s          

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~22s          

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/admae8w2_89i88tvv/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 38s

Computing TSS enrichment score
Found 7460 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines
Done Processing 28 million lines
Done Processing 29 million lines
                                                  
Done Processing 30 million lines
Done Processing 31 million lines
Done Processing 32 million lines
Done Processing 33 million lines
Done Processing 34 million lines
Done Processing 35 million lines
Done Processing 36 million lines
Done Processing 37 million lines

  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~02m 25s      
  |++++++++++                                        | 20% ~02m 19s      
  |+++++++++++++++                                   | 30% ~02m 26s      
  |++++++++++++++++++++                              | 40% ~01m 59s      
  |+++++++++++++++++++++++++                         | 50% ~01m 41s      
  |++++++++++++++++++++++++++++++                    | 60% ~01m 23s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 07s      
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~44s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~22s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 40s

Found 5572 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines

  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~02m 05s      
  |++++++++++                                        | 20% ~02m 06s      
  |+++++++++++++++                                   | 30% ~01m 57s      
  |++++++++++++++++++++                              | 40% ~01m 48s      
  |+++++++++++++++++++++++++                         | 50% ~01m 30s      
  |++++++++++++++++++++++++++++++                    | 60% ~01m 15s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~58s          
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~38s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~19s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 20s

Found 10837 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines
Done Processing 28 million lines
Done Processing 29 million lines
                                                  
Done Processing 30 million lines
Done Processing 31 million lines
Done Processing 32 million lines
Done Processing 33 million lines
Done Processing 34 million lines
Done Processing 35 million lines
Done Processing 36 million lines
Done Processing 37 million lines
Done Processing 38 million lines
Done Processing 39 million lines
                                                  
Done Processing 40 million lines
Done Processing 41 million lines
Done Processing 42 million lines
Done Processing 43 million lines
Done Processing 44 million lines
Done Processing 45 million lines
Done Processing 46 million lines
Done Processing 47 million lines
Done Processing 48 million lines
Done Processing 49 million lines
                                                  
Done Processing 50 million lines
Done Processing 51 million lines
Done Processing 52 million lines
Done Processing 53 million lines
Done Processing 54 million lines

  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~02m 41s      
  |++++++++++                                        | 20% ~02m 27s      
  |+++++++++++++++                                   | 30% ~02m 28s      
  |++++++++++++++++++++                              | 40% ~01m 59s      
  |+++++++++++++++++++++++++                         | 50% ~01m 40s      
  |++++++++++++++++++++++++++++++                    | 60% ~01m 20s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 03s      
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~42s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~21s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 27s

Found 6919 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines
Done Processing 28 million lines
Done Processing 29 million lines
                                                  
Done Processing 30 million lines
Done Processing 31 million lines
Done Processing 32 million lines
Done Processing 33 million lines
Done Processing 34 million lines

  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~03m 05s      
  |++++++++++                                        | 20% ~02m 26s      
  |+++++++++++++++                                   | 30% ~02m 15s      
  |++++++++++++++++++++                              | 40% ~02m 03s      
  |+++++++++++++++++++++++++                         | 50% ~01m 40s      
  |++++++++++++++++++++++++++++++                    | 60% ~01m 21s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~59s          
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~41s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~20s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 22s

Found 5530 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines

  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~02m 15s      
  |++++++++++                                        | 20% ~02m 26s      
  |+++++++++++++++                                   | 30% ~02m 19s      
  |++++++++++++++++++++                              | 40% ~01m 52s      
  |+++++++++++++++++++++++++                         | 50% ~01m 35s      
  |++++++++++++++++++++++++++++++                    | 60% ~01m 20s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 01s      
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~40s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~21s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 25s

Found 7910 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines
Done Processing 28 million lines
Done Processing 29 million lines
                                                  
Done Processing 30 million lines
Done Processing 31 million lines
Done Processing 32 million lines
Done Processing 33 million lines
Done Processing 34 million lines
Done Processing 35 million lines
Done Processing 36 million lines
Done Processing 37 million lines
Done Processing 38 million lines
Done Processing 39 million lines

  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~02m 11s      
  |++++++++++                                        | 20% ~02m 08s      
  |+++++++++++++++                                   | 30% ~02m 04s      
  |++++++++++++++++++++                              | 40% ~01m 46s      
  |+++++++++++++++++++++++++                         | 50% ~01m 31s      
  |++++++++++++++++++++++++++++++                    | 60% ~01m 15s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~58s          
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~39s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~19s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 17s

Found 5845 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines
Done Processing 28 million lines
Done Processing 29 million lines

  |                                                  | 0 % ~calculating  

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |+++++                                             | 10% ~02m 60s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |++++++++++                                        | 20% ~02m 44s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |+++++++++++++++                                   | 30% ~02m 32s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++                              | 40% ~02m 17s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |+++++++++++++++++++++++++                         | 50% ~01m 51s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++++++++++++                    | 60% ~01m 29s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 10s      

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++++++++++++++++++++++          | 80% ~46s          

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~23s          

[W::hts_idx_load2] The index file is older than the data file: /Users/mlromeror/Documents/Sample co7dzuup_xuczw9vc/data/Experiment/sr20954q_yiuuoxng/atac_fragments.tsv.gz.tbi

  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 58s

Found 7181 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines
Done Processing 28 million lines
Done Processing 29 million lines
                                                  
Done Processing 30 million lines
Done Processing 31 million lines
Done Processing 32 million lines
Done Processing 33 million lines
Done Processing 34 million lines
Done Processing 35 million lines

  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~02m 24s      
  |++++++++++                                        | 20% ~02m 22s      
  |+++++++++++++++                                   | 30% ~02m 12s      
  |++++++++++++++++++++                              | 40% ~01m 53s      
  |+++++++++++++++++++++++++                         | 50% ~01m 39s      
  |++++++++++++++++++++++++++++++                    | 60% ~01m 21s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 02s      
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~41s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~22s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 36s

Found 6103 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines
Done Processing 28 million lines
Done Processing 29 million lines
                                                  
Done Processing 30 million lines

  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~02m 03s      
  |++++++++++                                        | 20% ~02m 17s      
  |+++++++++++++++                                   | 30% ~02m 20s      
  |++++++++++++++++++++                              | 40% ~01m 59s      
  |+++++++++++++++++++++++++                         | 50% ~01m 41s      
  |++++++++++++++++++++++++++++++                    | 60% ~01m 23s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 02s      
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~43s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~21s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 28s

Found 7861 cell barcodes

Done Processing 1 million lines
Done Processing 2 million lines
Done Processing 3 million lines
Done Processing 4 million lines
Done Processing 5 million lines
Done Processing 6 million lines
Done Processing 7 million lines
Done Processing 8 million lines
Done Processing 9 million lines
                                                  
Done Processing 10 million lines
Done Processing 11 million lines
Done Processing 12 million lines
Done Processing 13 million lines
Done Processing 14 million lines
Done Processing 15 million lines
Done Processing 16 million lines
Done Processing 17 million lines
Done Processing 18 million lines
Done Processing 19 million lines
                                                  
Done Processing 20 million lines
Done Processing 21 million lines
Done Processing 22 million lines
Done Processing 23 million lines
Done Processing 24 million lines
Done Processing 25 million lines
Done Processing 26 million lines
Done Processing 27 million lines
Done Processing 28 million lines
Done Processing 29 million lines
                                                  
Done Processing 30 million lines
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print(pbmc)

[[1]]
An object of class Seurat 
110788 features across 5788 samples within 2 assays 
Active assay: ATAC (74187 features, 0 variable features)
 1 other assay present: RNA

[[2]]
An object of class Seurat 
91288 features across 7460 samples within 2 assays 
Active assay: ATAC (54687 features, 0 variable features)
 1 other assay present: RNA

[[3]]
An object of class Seurat 
152589 features across 5572 samples within 2 assays 
Active assay: ATAC (115988 features, 0 variable features)
 1 other assay present: RNA

[[4]]
An object of class Seurat 
87952 features across 10837 samples within 2 assays 
Active assay: ATAC (51351 features, 0 variable features)
 1 other assay present: RNA

[[5]]
An object of class Seurat 
160217 features across 6919 samples within 2 assays 
Active assay: ATAC (123616 features, 0 variable features)
 1 other assay present: RNA

[[6]]
An object of class Seurat 
169041 features across 5530 samples within 2 assays 
Active assay: ATAC (132440 features, 0 variable features)
 1 other assay present: RNA

[[7]]
An object of class Seurat 
90100 features across 7910 samples within 2 assays 
Active assay: ATAC (53499 features, 0 variable features)
 1 other assay present: RNA

[[8]]
An object of class Seurat 
110486 features across 5845 samples within 2 assays 
Active assay: ATAC (73885 features, 0 variable features)
 1 other assay present: RNA

[[9]]
An object of class Seurat 
144601 features across 7181 samples within 2 assays 
Active assay: ATAC (108000 features, 0 variable features)
 1 other assay present: RNA

[[10]]
An object of class Seurat 
156602 features across 6103 samples within 2 assays 
Active assay: ATAC (120001 features, 0 variable features)
 1 other assay present: RNA

[[11]]
An object of class Seurat 
134743 features across 7861 samples within 2 assays 
Active assay: ATAC (98142 features, 0 variable features)
 1 other assay present: RNA

#List of all the fragment path of each sample 

SeuratObjetList<- function(path){
  fragpath <-list.files(path=path,
                      pattern=".tsv.gz$", recursive = T, full.names = TRUE)
  #list of all the sample names
  sample.name<- sub(".*Experiment\\/(.*)\\/filtered_feature_bc_matrix.h5","\\1", fragpath, perl = TRUE )

  # get gene annotations for hg38
  annotation <- GetGRangesFromEnsDb(ensdb = EnsDb.Hsapiens.v86)
  seqlevelsStyle(annotation) <- "UCSC"
  genome(annotation) <- "hg38"

# create a Seurat object containing the RNA adata
  
multiome = make.multiome.List(path)

pbmc_SeuratObjects = lapply(multiome, function(x) CreateSeuratObject(counts = x$`Gene Expression`,  assay= "RNA"))
return(pbmc_SeuratObjects)
}

pbmc_SeuratObjects<-SeuratObjetList("data/Experiment")




# load the RNA and ATAC data

#counts_qmzb59ew_t11l8dzm <- Read10X_h5(filename = "data/Experiments/filtered_feature_bc_matrix.h5")

# create ATAC assay and add it to the object
pbmc[["ATAC"]] <- CreateChromatinAssay(
  counts = counts$Peaks,
  sep = c(":", "-"),
  fragments = fragpath,
  annotation = annotation
)

for (i in seq_along(pbmc_SeuratObjects)){
  pbmc_SeuratObjects[[i]["ATAC"]]<- assign(pbmc_SeuratObjects[["ATAC"]], 
                                                CreateChromatinAssay(counts = multiome[[i]]$Peaks,
                                                                     sep = c(":", "-"),
                                                                     fragments = fragpath[[i]],
                                                                     annotation = annotation), envir = .GlobalEnv
  )
}

pbmc

Quality control

pbmc <- NucleosomeSignal(pbmc2)

Found 5572 cell barcodes

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pbmc <- NucleosomeSignal(pbmc2)

Found 5572 cell barcodes

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pbmc <- TSSEnrichment(pbmc)

Extracting TSS positions
Extracting fragments at TSSs

  |                                                  | 0 % ~calculating  
  |+++++                                             | 10% ~02m 54s      
  |++++++++++                                        | 20% ~02m 55s      
  |+++++++++++++++                                   | 30% ~02m 49s      
  |++++++++++++++++++++                              | 40% ~02m 17s      
  |+++++++++++++++++++++++++                         | 50% ~01m 51s      
  |++++++++++++++++++++++++++++++                    | 60% ~01m 30s      
  |+++++++++++++++++++++++++++++++++++               | 70% ~01m 07s      
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~46s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~22s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03m 42s

Computing TSS enrichment score

# filter out low quality cells
pbmc2 <- subset(
  x = pbmc2,
  subset = nCount_ATAC < 100000 &
    nCount_RNA < 25000 &
    nCount_ATAC > 1000 &
    nCount_RNA > 1000 &
    nucleosome_signal < 2 &
    TSS.enrichment > 1
)
pbmc2

data_path <- file.path(Sys.getenv("data/co7dzuup_xuczw9vc"), "data/co7dzuup_xuczw9vc")
save_path <- file.path(Sys.getenv("data/co7dzuup_xuczw9vc/"), "seu")
data_path
save_path
dataset <- "filtered_feature_bc_matrix"

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